Ralstonia ahl-acylase gene

ABSTRACT

This invention provides a gene, qsbA, which encodes a protein useful for inactivating certain bacterial quorum-sensing signal molecules (N-acyl homoserine lactones) which participate in bacterial virulence and biofilm differentiation pathways. This gene was isolated from  Ralstonia  sp., strain XJ12B. The invention also provides the QsbA protein, which possesses N-acyl homoserine lactone inactivating activity.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of molecular biology. Inparticular, the invention relates to an N-acyl homoserine lactoneacylase gene from Ralstonia sp. XJ12B.

2. Description of the Background Art

N-acyl homoserine lactones (AHLs), also known as autoinducers, arewidely used quorum sensing signal molecules in many Gram-negativebacteria. These compounds regulate certain classes of target genes inbacteria, such as virulence genes or biofilm differentiation genes.Generally, quorum sensing molecules are highly conserved and share anidentical homoserine lactone moiety. The length and structure of theiracyl side chains are different, however. Although the target genesregulated by AHLs in different bacteria species are varied, basicmechanisms of AHL biosynthesis and gene regulation are conserved amongdifferent bacterial species.

The general feature of AHL-mediated gene regulation is that it is cellpopulation dependent (quorum sensing). Bacteria secrete AHLs into theenvironment; extracellular concentration of AHLs increases as bacterialcell populations grow. When AHL accumulates to a threshold extracellularconcentration, the expression of certain sets of target genes aretriggered in the bacteria.

Bacteria using these signals release, detect and respond to theaccumulation of AHL signal molecules for synchronizing expression of aparticular sets of genes and coordinating cellular activities within thebacterial cell population. AHLs are involved in regulation of a range ofbiological functions, including bioluminescence in Vibrio species (13,4), Ti plasmid conjugal transfer in Agrobacterium tumefaciens (31),induction of virulence genes in Burkhholderia cepacia, Erwiniacarotovora, Erw. chrysanthemi, Erw. stewartii, Pseudomonas aeruginosa,and Xenorhabdus nematophilus (3, 6, 12, 17, 19, 22, 23, 24, 26),regulation of antibiotic production in P. aureofaciens and Erw.carotovora (6, 26), swarming motility in Serratia liquifaciens (14) andbiofilm formation in P. fluorescens and P. aeruginosa (1, 8). In manyother bacterial species the relevant biological functions controlled byAHLs remain to be investigated (2, 5, 11).

A number of plant, animal and human bacterial pathogens use AHLquorum-sensing signals to regulate expression of pathogenic genes andaid in the formation of biofilms. Therefore, AHL quorum-sensing signalmolecules are group of molecular targets for genetic and chemicalmanipulations since disruption of these signaling mechanisms can preventor reduce the ability of these bacteria to infect plant and animaltissues or to form biofilms.)

The gene encoding an AHL-inactivation enzyme (AiiA) from a Gram-positivebacterium (Bacillus strain 240B1) has been cloned (9). AiiA (also knownas AHL-lactonase) inactivates AHL activity by hydrolyzing the lactonebond of AHLs (10). Expression of aiiA in transformed Erw. carotovora (apathogenic strain which causes soft rot disease in many plants)significantly reduces the release of AHL, decreases extracellularpectrolytic enzyme activities, and attenuates pathogenicity on potato,eggplant, Chinese-cabbage, carrot, celery, cauliflower, and tobacco (9).Transgenic plants expressing AHL-lactonase showed a significantlyenhanced resistance to Erw. carotovora infection and delayed developmentof soft rot symptoms (10). AHL-inactivation mechanisms appear to bewidely distributed. For example, a bacterial isolate of Variovoraxparadoxus was reported to use AHL molecules as its energy and nitrogensources, indicating the possible presence of AHL-degrading enzymes (18).

Further methods to counteract AHL-mediated plant, animal and humandisease and plant pathogen virulence by interfering with bacterialintercellular communication would be highly desirable.

SUMMARY OF THE INVENTION

Accordingly, in this study, the cloning and characterization of a geneencoding an AHL-acylase from a bacterial isolate Ralstonia sp. JX12B isreported.

In one embodiment, the invention provides a composition of matter whichcomprises a nucleic acid according to SEQ ID NO: 1. In anotherembodiment, the invention provides a composition of matter whichcomprises a nucleic acid selected from the group consisting ofnucleotides 1234-3618 of SEQ ID NO: 1, a fragment thereof and asubstantially homologous variant thereof.

In yet a further embodiment, the invention provides a nucleic acidaccording to claim 2 which comprises nucleotides 1234-3618 of SEQ ID NO:1.

In yet a further embodiment, the invention provides a composition ofmatter which comprises a peptidic sequence selected from the groupconsisting of a peptidic sequence according to SEQ ID NO: 2, a fragmentthereof and a substantially homologous variant thereof.

In yet a further embodiment, the invention provides a composition ofmatter which comprises a peptidic sequence encoded by a nucleic acidselected from the group consisting of nucleotides 1234-3618 of SEQ IDNO: 1, a fragment thereof and a substantially homologous variantthereof.

In yet a further embodiment, the invention provides a composition ofmatter which comprises a peptidic sequence selected from the groupconsisting of SEQ ID NO: 2, a fragment thereof, a subunit thereof and asubstantially homologous variant thereof, such as a peptidic sequenceaccording to SEQ ID NO: 2, a peptidic sequence comprising amino acids36-217 233-794[?] of SEQ ID NO: 2 or a peptidic sequence comprisingamino acids 233-794 of SEQ ID NO: 2.

In yet a further embodiment, the invention provides a composition ofmatter as described above which inactivates AHL.

In yet a further embodiment, the invention provides a method ofmodulating AHL signaling activity which comprises contacting said AHLwith a composition of matter as described above.

In yet a further embodiment, the invention provides a transgenic plantor non-human mammal harboring a nucleic acid as described above.

In yet a further embodiment, the invention provides a method ofcontrolling a bacterial disease in a mammal which comprisesadministering to said mammal a composition of matter as described above,wherein the expression of pathogenic genes of said bacteria areregulated by AHL signals.

In yet a further embodiment, the invention provides a method ofcontrolling a bacterial disease in a plant which comprises administeringto said plant a composition of matter as described above, wherein theexpression of pathogenic genes of said bacteria are regulated by AHLsignals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing AHL inactivation bioassay results forbacterial cultures and bacterial proteins from the indicated bacterialclones. FIG. 1A shows the results of a bioassay with bacterial culturesof E. coli DH5α strains 13H10 (slice 1), 2B10 (slice 2), MUB3 (slice 3),MUC6 (slice 4), GST-QsbA (slice 5) and GST (slice 6), which containplasmid clones or constructs p13H10, p2B10, pMUB3, pMUC6, pGST-QsbA, andpGST, respectively. FIG. 1B shows results for bioassay of the indicatedbacterial proteins GST-QsbA and GST.

FIG. 2 is a graph showing the temperature and pH optimum profiles of AHLacylase.

FIG. 3 is a graph showing the time course of OOHL inactivation by thepurified AHL-acylase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A bacterial isolate of Ralstonia sp. XJ12B from a biofilm sample in awater treatment system was found to enzymatically inactivate AHLs,bacterial quorum-sensing molecules, in a bioassay using Agrobacteriumtumefaciens strain Nt1 (traR; tra:lacZ749) as an indicator for AHLactivity. The gene encoding the protein exhibiting this enzyme activityfor AHL inactivation (qsbA) was cloned from a bacterial strain isolatedfrom the biofilm sample and found to encode a peptide of 794 aminoacids.

Bacterial cultures and bacterial proteins were assayed for the abilityto inactivate AHL using Agrobacterium tumefaciens indicator cells. Atumefaciens was cultured at 28° C. in MM medium as described in Zhang etal. (31). The bacteria or protein to be assayed is first mixed with anAHL substrate, for example N-β-oxooctanoyl-L-homoserine lactone (OOHL),and the reaction (inactivation of the AHL) is allowed to proceed. If AHLinactivation activity is present in the sample (i.e. the AHL has beencleaved and inactivated), then the inactivated AHL products fail totrigger the expression of lacZ reporter gene which is under the controlof a TraR-dependent promoter. The strain A. Tumefaciens NT1 hosting thelacZ reporter system therefore does not turn blue in the presence ofsubstrate 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal). SeeExample 2 for details of the bioassay. Any AHL may be used in the assay,as desired. Of course, any suitable assay for cleavage of AHL, includingtraditional in vitro enzyme assays may be used to detect the AHLinactivation activity. Those of skill in the art are able to modify ordevise assays to detect and/or quantitate AHL inactivation.

Escherichia coli strain DH5a was used as a host for DNA manipulation.Both Ralstonia sp. and E. coli were cultured in LB medium (tryptone, 10g/L, yeast extract, 5 g/L, and NaCl, 10 g/L, pH 7.0) at 37° C.Appropriate antibiotics were added when necessary at the followingconcentrations: ampicillin, 100 μg/ml; tetracycline, 10 μg/ml; andkanamycin, 20 μg/ml.

The gene encoding the protein responsible-for the detected AHLinactivation was isolated using a cosmid library of 1600 clones with thegenomic DNA of Ralstonia sp. strain XJ12B, constructed in E. coli. E.coli transfectants were screened for the ability to inactivate AHL. Oneclone, p13H10, was found to inactivate AHL. Cosmid DNA from p13H10 wasdigested, fused into a cloning vector, ligated and transformed into E.coli. The E. coli clones again were assayed for AHL inactivatingactivity. One clone, containing a 4 kb insert, had AHL inactivationactivity.

Plasmids were subsequently purified for sequencing. The 4 kb fragmentfrom clone p2B10 was completely sequenced according to known methodsusing ABI Prism dRhodamine Terminator Cycle Sequencing Ready ReactionKit (Perkin-Elmer Applied Biosystems). See Table I, below. The sequencecontained an open reading frame of 2385 nucleotides which was the AHLinactivation gene, qsbA, encoding a predicted polypeptide of 794 aminoacids (85,373 Daltons). TABLE I QsbA Gene (Ralstonia sp.) NucleotideSequence (SEQ ID NO: 1). gtttgggaaagtgggnagcgcgctgtgcag 90cgccccgcccctcagccgcgcagctcggcg cgcaccgaatgcgcgcgccggtgggcgcccggcggctggccggtgtggcgccggatcagg 180 cgccggaaggcggacatgtcgtgataaccgcactgttcggcgattgccgtcaggctcagc gtgctgacttccagcaggtggcaggcgcgc 270tccacgcgcagccggtgcagcaattgcagc ggcgaggtgcccagggtcttggtgaaatgccgcagcagcgtgcgctcgctggtcgaggcg 360 gcggcggccagcttggccaggtcgaacggctcgtgcaggtgctgctgcaggtagcgccgc gcccgcagtaccacgctggtgcggatggcg 450ggcttgctgcgcagccagatggcggtggac tcaccgcgcgacgggtggtcgagcacggcctggccgagggtgcgtgccagccgggtgtcg 540 gccaggcggccgaccaggcgctgcgtgagcgccacgccgtgctccatcgcgcgcgccgtc agcacgttgccgctgctgacgatggcctgc 630tccgccaccaccttcagctgcgggtagttg ccgtgcagccagccggcgatcagccacgtcaccgtcaagcgccggccggcgggcagcgcg 720 ccggccagcagcgccacgccggtgaaggacgaggccaccaagcctgccggcgtccaggta gcgccggatggtggcgcgttcccactccag 810cagggccaggcgctgctccagcgtgctgat gtggtcgaaatgcaggggcgggacgaccagcgcgtcgcccagcgcggcgtcgcccgccgg 900 cagcggctggcagcggcaggccagggtctcggcggcggcctgccagcgggccgggtcgcg cgcgaccagccgccacccgaacaccgggct 990ggcggcatcggcacgcttgccggcatgcat ggaggcgagcgcattggccacgccgagggtgtcggcgacggtcgccagcgtggagaggcc 1080 ggcgtcgggaaaggtcagcaggtcgatgtcggcatccgcaaagtataggggaggcgggcg gaggcctcctgcgtggcgggattgacccca 1170actctggcgggaatacctctttcctccggg cgggccccagtcgacgatacggcggtggctgcgcctgcgcgccgccgcaagactagagcg 1260 acacaagacaagaccgacaacaggagacaacgcATGATGCAGGGATTCGCGCTGCGCGGC ACGCTCGCCATGGCCGCGCTCGCGGCGCTG 1350GCCGGCTGCGCCAGTTCCACCGATGGCCGC TGGGGGTCGCTCAGCGACACCGGCCTGTCCGCCGAGATCCGCCGCACCGGCTTCGGCATT 1440 CCGCACATCCGCGCCAACGACTACGCCAGCCTCGGCTATGGCATGGCCTATGCCTACGCG CAGGACAACCTGTGCCTGCTGGCCGACCAG 1530GTGGTCACCGTCAACGGCGAGCGCTCGAAG ACCTTCGGGCCCGAGGGCACCGTGACGGTCTCGTTCAAGCCGATCCCCAACCTGCAGTCG 1620 GACGCCTTCTTCAAGGGCATCTTCGACGAGGACGGCCTGCGCGCCGGTTATGCGCAGATG TCGCCCGAGGCGCGCGAGCTGCTGCGCGGC 1710TACATCGCCGGCTTCAACCGCTATCTCAAG GACACGCCGCCCGCCAACTTCCCGGCCGCCTGCCGCAATGCCGCCTGGGTGCGTCCGCTC 1800 ACGCTGGGCGACATGATGCGCATGGGCGAAGAGAAGGCGATCCAGGCCAGCGCCGGCGCC ATGCTGGCGGGCATCGTCGCCGCGCAGCCG 1890CCGGGCCGCACGCCGGTGGCCGAGCGCGAG ATTCCGCCGCAGGCCGTCGACACCGTGGCGCTGGACCGCGAACTGCAGCTGCGCGACATG 1980 CCGATCGGCTCCAACGGCTGGGCCTTCGGCGCTGACGCCACCGCCAACCGGCGCGGCGTG CTGCTCGGCAATCCGCACTTCCCGTGGACG 2070ACCACCAACCGCTTCTACCAGGTCCACCTG ACGGTGCCCGGCAAGCTCGACGTGATGGGCGCCTCGATCGCGGCCTTCCCGGTGGTGAGC 2160 ATCGGCTTCAACAAGGACGTGGCGTGGACGCACACCGTCTCCACCGGCCGCCGCTTCACC TTGTTCGAACTGAAGCTGGCCGAAGGCGAC 2250CCGACCACCTACCTGGTCGACGGCACGCCG CACAAGATGACCACCCGCACGGTCGCCTTCGACGTGAAGCTGCCGGACGGCCGCCTCGAG 2340 CGCCGCACGCACACCTTCTACGACACCATCTACGGCCCGGTGCTGTCGATGCCGAGCGGC GGCATGCCGTGGACCACGCAGAAGGCCTAC 2430GCCCTGCGCGACGCCAACCGCAACAACACG CGCTCGGTCGACAGCTGGCTGCATATCGGGCAGGCCCGGGACGTGGCCGGCATCCGCCAG 2520 GCCATCGGCAACCTGGGCATTCCCTGGGTCAACACCATCGCCACCGACCGCAACGGCCGC GCGCTGTTCGCCGACGTGTCGACCACGCCG 2610GACGTGCCGGCCGCGGAGCTCCAGCGCTGT GCCCCGTCGCCGCTGGCCGGCAAACTCTTCAAGGACGCGGGCCTGGTGCTGCTCGACGGC 2700 TCGCGCGGCACCTGCAACTGGCAGGTCGATCCGGCTTCGCCGGTACCCGGGCTGGTGGCG CCCGCGCGCATGCCGGTGCTCGAGCGCGAC 2790GACTACGTCGCCAACAGCAATGACAGCTCC TGGCTGACCAACCCCGCGCAAAAGCTGACCGGCTTCTCGCCGGTGATGGGCTCGGTCGAC 2880 GTACCGCAGCGGCTGCGCACGCGCATCGGCCTGATCGAGATCGGCCGCCGCCTGGCCGGC ACCGACGGACTGCCCGGCAACCGCATCGAT 2970CTGCCGAACCTGCAGGCGATGATCTTCAGC AATGCCAACCTGGCGGGACAACTGGTGCTGGGCGACCTGCTCGCGGCATGCAAGGCCACG 3060 CCGGCCCCGGATGCCGACGTGCGCGACGGCTGCGCCGCCCTCGGCCAGTGGAACCGCACC AGCAACGCCGACGCCCGCGCCGCGCACCTG 3150TTCCGCGAGTTCTGGATGCGCGCCAAGGAC ATCGCGCAGGTGCACGCCGTCGAGTTCGACCCGGCCGACCCGGTCCACACGCCGCGCGGC 3240 CTGCGCATGAACGACGCGACGGTACGCACGGCGGTGTTCAAGGCGCTGAAGGAAGCCGTG GGCGCGGTGCGCAAGGCGGGCTTCGCGCTG 3330GATGCGCCGCTGGGCACGGTACAGGCCGCG CACGCACCGGACGGCTCCATCGCCCTGCACGGCGGCGAGGAATACGAAGGCGTGCTCAAC 3420 AAGCTGCAAACCCTGCCGATCGGGCCGAAGGGGCTGCCGGTGTATTTCGGCACCAGCTAC ATCCAGACCGTGACCTTCGACGACCAGGGC 3510CCGGTCGCCGACGCCATCCTCACCTACGGC GAATCGACCGACCACGCCTCGCCGCACGCGTTCGACCAGATGCGTGCGTACTCGGGCAAG 3600 CACTGGAACCGGCTGCCGTTCTCCGAAGCGGCCATCGCGGCCGATCCGGCGCTGAAGGTG ATGCGGTTGTCGCAGTGAgggctgccggtg 3690cctggaaaaacgccccgcttgtgcggggcg tttttttgccagtgtgaatggctcaatcgtgttggaaaccgcatccggacatgactgtat 3743 tgtgactctgcctgtgtccgtgtThe predicted open reading frame of the qsbA gene is shown in upper caseletters with the start codon and stop codon in bold. A putative ribosomebinding site (AGGAGA) is underlined.

Sequence analysis of this peptide indicated that QsbA did not have anysignificant homology with the known AHL-lactonase quorum-sensingmolecule inactivator encoded by the aiiA gene from Bacillus sp. 240B1,however the deduced peptide sequence was typical of the primarystructure of aculeacin A acylases (AACs) and penicillin G acylases, withsignal peptide-α subunit-spacer-β subunit organization (16, 30). TheRalstonia sequence shares substantial identity with AACs fromDeinococcus radiodurans strain R1, Actinoplanes utahensis and a putativeacylase from Pseudomonas aeruginosa, all of which catalyze deacylationof their substrates. These AAC genes are translated as single precursorpolypeptide and then processed to the active form, which has twosubunits. Aculeacin A is an echinocandin-type antifungal antibiotic witha long fatty side chain. Aculeacin A acylases purified from A. utahensiscatalyze the hydrolysis of the amide bond on the palmitoyl side chain ofaculeacin A (29). The primary structure of the protein, as well asenzyme activity analysis with different substrates, discussed below,therefore indicates that qsbA encodes an AHL-acylase which cleaves theamide linkage between the acyl side chain and the homoserine lactonemoiety of AHLs.

The presumed α and β subunits of QsbA are located at amino acidpositions 36-217 and 233-794, respectively, of SEQ ID NO: 2, with a 15amino acid spacer between them, as determined by alignment with thepeptide sequences from D. radiodurans strain R1, A. utahensis and P.Aeruginosa. See Table II. TABLE II Aligned Amino Acid Sequences of QsbAfrom Ralstonia sp. XJ12B (SEQ ID NO: 2), D. radiodurans strain R1acylase (SEQ ID NO: 3), A. utahensis acylase (SEQ ID NO: 4) and P.aeruginosa acylase (SEQ ID NO: 5). R. spMMQGF---ALRGTLAMAALAALAGCA-----SSTDGRWGSLSDTGLSAEIRRTGFGIPHIRANDYASLGYGMAYAYAQDN72 D. radMSR-----SPFSSVSLPARLLLGSLL-----LGPLMLGGAASAQTYQVQIQRTAHGIPHIQASDLGGIGYGVGYSYAQDN70 A. utaMTSSY---MRLKAAAIAFGVIVATAA-----VPSPAS-GREHDGGYAALIRRASYGVPHITADDFGSLGFGVGYVQAEDN71 P. aerMSRPFRPPLCRETTSMGMRTVLTGLAGMLLGSMMPVQADMPRPTGLAADIRWTAYGVPHIRAKDERGLGYGIGYAYARDN80*           : ::     :                .        . *: :..*:*** *.*  .:*:*:.*  *.**R. spLCLLADQVVTVNGERSKTFGPEGTVTVSFKPIPNLQSDAFFKGIFDEDGLRAGYAQMSPEARELLRGYIAGFNRYLKDTP152 D. radLCLLADQVMTVRGERSKFLGAEGKTVVGFQPVNNLDSDVFFKTVIEPGRLQAGYRDQ-PQILALMRGYVAGVNRYLRDTP149 A. utaICVIAESVVTANGERSRWFGATGPDDADVRTTSSTQAIDDRVAERLLEGPRDGVRAPCDDVRDQMRGFVAGYNHFLRRTG151 P. aerACLLAEEIVTARGERARYFGSEGKSSAELD---NLPSDIFYAWLNQPEALQAFWQAQTPAVRQLLEGYAAGFNRFLREAD157  *::*:.::*..***.:: :*. *   . .    .  : R. spPANFP-AACRNAAWVRPLTLGDMMRMGEEKAIQASAGAMLAGIVAAQPPGRTPVAEREIPPQAVDTVALDRELQLRDMPI231 D. radPEQWP-SACRNADWVRPLTELDVMRLGEEKAIQASAGAMVSAITSARPPQ----AGASTAAPRPDLAAFNRQYRFNDLPI224 A. utaVHRLTDPACRGKAWVRPLSEIDLWRTSWDSMVRAGSGALLDGIVAATPPT---AAGPASAPEAPDAAAIAAALDGTSAGI228 P. aerGKTTS---CLGQPWLRAIATDDLLRLTRRLLVEGGVGQFADALVAAAPPG----AEKVALSGEQAFQVAEQRRQRFRLER230     .   * .  *:*.::  *: *      :... * :  .:.:* **     *     .      .                                                                 ↑--------R. spGSNGWAFGADATANRRGVLLGNPHFPWTTTNRFYQVHLTVPGKLDVMGASIAAFPVVSIGFNKDVAWTHTVSTGRRFTLF311 D. radGSNGWAFGSEATTNGRGLLLGNPHFPWETSNRFYQLHLTLPGQFDVMGASLGGMPVVNIGFNQDVAWTHTVSTDKRFTLA284 A. utaGSNAYGLGAQATVNGSGMVLANPHFPWQGAERFYRMHLKVPGRYDVEGAALIGDPIIEIGHNRTVAWSHTVSTARRFVWH288 P. aerGSNAIAVGSERSADGKGMLLANPHFPWNGAMRFYQMHLTIPGRLDVMGASLPGLPVVNIGFSRHLAWTHTVDTSSHFTLY290***. ..*:: :.:  *::*.*****  : ***::**.:**: ** **:: . *::.**..: :**:***.*  :*.R. spELKLAEGDPTTYLVDGTPHKMTTRTVAFDVKLPDGRLERRTHTFYDTIYGPVLSMPSGGMPWTTQKAYALRDANRNNTRS391 D. radALTLVPGDPLSYVKDGQQRRLQRRTAVIEVKTANG-PRLHTRTVYFTPEGPLVNLPAAGLTWTPQYAFALRDANRNNTRM383 A. utaRLSLVPGDPTSYYVDGRPERMRARTVTVQTGSG-----PVSRTFHDTRYGPVAVVP-GTFDWTPATAYAITDVNAGNNRA383 P. aerRLALDPKDPRRYLVDGRSLPLEEKSVAIEVRGADGKLSRVEHKVYQSIYGPLVVWP-GKLDWNRSEAYALRDANLENTRV390 * *   **  *  **    :  ::...:.           :..: :  **:   * . : *.   *:*: *.*  *.*R. spVDSWLHIGQARDVAGIRQAIG-NLGIPWVNTIATDRNGRALFADVSTTPDVPAAELQRCAPSPLAGKLFKDAGLVLLDGS470 D. radLATWLGFAGAKSVRDIRASLN-VQGIPWVNTIAADRAGSALYADISSSPNVSAAQQQACTPPPLA-PLFPAAGLAVLDGS461 A. utaFDGWLRMGQAKDVRALKAVLDRHQFLPWVNVIAADARGEALYGDHSVVPRVTGALAAACIPAPFQ-PLYASSGQAVLDGS461 P. aerLQQWYSINQASDVADLRRRVEALQGIPWVNTLAADEQGNALYMNQSVVPYLKPELIPACAIPQLV-----AEGLPALQGQ464.  *  :  * .*  ::  :     :****.:*:*  * **: : *  * :       *  . :        *   *:*.R. spRGTCNWQVDPASPVPGLVAPARMPVLERDDYVANSNDSSWLTNPAQKLTGFSPVMGSVDVPQRLRTRIGLIEIGRRLAGT550 D. radHSACDWKTDPASRVPGLRAPDKMPVLIRQDFVANSNNSAWLANPAAPQTGLDPLVGEVNAPQSPRTRMGLLEIGRRLSGT541 A. utaRSDCALGADPDAAVPGILGPASLPVRFRDDYVTNSNDSHWLASPAAPLEGFPRILGNERTPRSLRTRLGLDQIQQRLAGT541 P. aerDSRCAWSRDPAAAQAGITPAAQLPVLLRRDFVQNSNDSAWLTNPASPLQGFSPLVSQE-KPIGPRARYALSRLQGKQP--543  . *    ** :  .*:  .  :**  * *:* ***:* **:.**    *:  ::.. R. spDGLPGNRIDLPNLQAMIFSNANLAGQLVLGDLLAACKATPAPDAD------VRDGCAALGQWNRTSNADA-RAAHLFREF630 D. radDGLPGRTFDIPTLQATLLRESNLTGEMYAADAAKLCQS--AGGAE------LQPACNALAAWDRRSSQES-RGAALWREF619 A. utaDGLPGKGFTTARLWQVMFGNRMHGAELVRDDLVALCRRQPTATASNGAIVDLTAACTALSRFDERADLDS-RGAHLFTEF621 P. aer-------LEAKTLEEMVTANHVFSADQVLPDLLRLCRDN-QGEKS------LARACAALAQWDRGANLDSGSGFVYFQRF613       :    *   :  :    .:    *    *:       .      :  .* **. ::. :. ::  .   : .*R. spWMRAKDIAQVHAVEFDPADPVHTPRGLR-MNDATVRTAVFKALKEAVGAVRKAGFALDAPLGTVQAAHAPDGSIALHGGE702 D. radWRRARAIPNVYAVPFDPADPVNTPRGLN-TADPAAQTALLGALREAAAALTAAGIPFDAPLGEVQGVVRGGDFISLPGGA691 A. utaLAGG----IRFADTFEVTDPVRTPAPFWNTTDPRVRTALADACNGSPASPSTR------SVGDIHTDSRGERRIPIHGGR690 P. aerMQRFAELDGAWKEPFDAQRPLDTPQGIA-LDRPQVATQVRQALADAAAEVEKSGIPDGARWGDLQVSTRGQERIAIPGGD686              *:   *: **  :     . . * :  *   : .             * ::        *.: **R. spEYEGVLNKLQTLPIGPKGLPVYFG--TSYIQTVTFDDQGPVADAILTYGESTDHASPHAFDQMRAYSGKHWNRLPFSEAA780 D. radEFEGVLDKIDFNPLAPGGYRGVVGNASSYIQTVGFTDSGVQAEAVLTYSQSSNPESPYFSDQTRLFSRSEWVKLPFTQPE771 A. utaGEAGTFNVITNPLVPGVGYPQVVHG-TSFVMAVELGPHGPSGRQILTYAQSTNPNSPWYADQTVLYSRKGWDTIKYTEAQ769 P. aerGHFGVYNAIQS--VRKGDHLEVVGG-TSYIQLVTFPEEGPKARGLLAFSQSSDPRSPHYRDQTELFSRQQWQTLPFSDRQ763   *. : :    :   .    .   :*::  * :   *  .  :*::.:*::  **   **   :* . *  : :::R. sp IAADPALKVMRLSQ--- 794 D. rad IEADPTRTVVQLSE--- 785 A. utaIAADPNLRVYRVAQRGR 786 P. aer IDADPQLQRLSIRE--- 777 * ***      : :* = identical residues, : = conserved substitutions; . = semi-conservedsubstitutions; ↑ = post-translational processing sites for signalpeptide and subunits; - = spacers.

The coding region of the qsbA gene was amplified by PCR. The amplifiedPCR products were digested, fused in-frame to the coding sequence of theglutathione S-transferase (GST) gene and expressed in E. coli. Proteinextracts from the recombinant E. coli cells were assayed for the abilityto inactivate AHL. Protein from E. coli expressing GST alone served as acontrol. The results demonstrated that GST-QsbA fusion proteineffectively eliminated AHL activity. See FIG. 1B.

The substrate specificity of QsbA was determined by assaying totalsoluble protein extracted from the recombinant E. coli (pGST-QsbA) forinactivation of AHLs using substrates with acyl chains of differinglengths. QsbA was able to completely inactivate 3-oxo group acyl-HSLshaving acyl chains of 8, 10 and 12 carbons. QsbA also stronglyinactivated methylene group acyl-HSLs having acyl chains of 8 and 10carbons. QsbA also inactivated the butyl and hexyl esters ofN-β-octanoyl-L-homoserine, whereas the AHL-lactonase encoded by aiiA wasunable to inactivate them. The substrate specificity data indicate thatQsbA is an AHL-acylase.

QsbA and qsbA provide new tools for down regulation of AHL-mediatedbiological activities, such as the expression of virulence genes andbiofilm differentiation in pathogenic bacteria, both in vitro and invivo. The qsbA gene, which encodes the AHL inactivation enzyme (QsbA),or a functional fragment, subunit or substantially homologous variantthereof, may be introduced into a plant genome to produce a geneticallymodified plant with the ability to quench pathogen quorum-sensingsignaling. Transgenic plants expressing an enzyme that inactivates AHLscan exhibit a significantly enhanced resistance to infection bybacterial pathogens, even when bacteria are present in highconcentrations.

Methods of genetic manipulation and transformation of plant cells arewell known in the art, as are methods of regenerating fertile, viabletransformed plants. In general, any method of cloning the coding regionof qsbA or a functional fragment or substantially homologous variantthereof into a suitable expression vector may be used. It is convenientto ligate the qsbA coding region into a vector, followed by ligationinto a plant transformation vector, however those of skill are wellaware of alternative methods to achieve the same results. Any suitableplant transformation vector may be used. The vector contains the qsbAgene, or a functional fragment, subunit or substantially homologousvariant thereof, so long as expression of the gene results in a QsbAprotein or functional fragment, subunit or variant thereof whichinactivates AHL.

A functional promoter preferably controls expression. Many suitablepromoters are known in the art, such that a convenient promoter mayeasily be selected by a skilled artisan depending on the expressionsystem being used. Such selection of a suitable promoter to achieve thedesired level of translational expression is considered routine in theart. For example, it is advantageous to optimize qsbA expression bymodification of codon usage and coupling to a strong promoter such asthe double 35S promoter.

A suitable marker gene, such as kanamycin resistance, green fluorescentprotein or any other convenient marker is advantageously used.Variations of the commonly used and well known methods for transformingplants with a gene, are well within the skill of the ordinary artisan ingenetic manipulation of plants. Expression constructs may contain asignal sequence to direct secretion of the expressed QsbA protein, ormay lack such a sequence, as desired. The plant transformation vectorscontaining the qsbA gene and a marker gene may be used to transformplant cells using Agrobacterium-mediated transformation.Agrobacterium-mediated transformation is conveniently used to transformplants with the qsbA gene, however any suitable method known in the artmay be used, depending on the plant being transformed. For example,certain monocotyledonous plants are more efficiently transformed usingother methods such as microprojectile bombardment, vacuum filtration orany other method known in the art to introduce and integrate DNAplasmids or fragments into the plant genome. Those of skill in the artare familiar with means to transform gymnosperms, monocots and dicots.All of these methods known in the art are contemplated for use with thisinvention.

After selection for transformants carrying the qsbA gene, transgenicplants may be regenerated according to known methods in the art. Plantsselected for a marker gene, for example kanamycin resistance, may beassayed, for example by PCR and DNA gel blot to determine how manycopies of the qsbA gene are present in the plant tissue. Any suitablemethod known in the art is contemplated for use with the gene of thisinvention. QsbA enzyme activity may be detected in transgenic plantstransformed with the qsbA gene by the bioassay method described inExample 2 or by any convenient method.

By “functional fragment, subunit or substantially homologous variantthereof,” when referring to a qsbA nucleotide sequence, it is meant anyfragment, subunit, variant or homologous sequence of qsbA (nucleotides1234-3618 of SEQ ID NO: 1) which encodes a protein or peptide sequencecapable of inactivating N-acyl homoserine lactones. “Substantiallyhomologous variants” of a nucleotide sequence generally are those thecomplement of which hybridizes with qsbA under stringent or highlystringent conditions, for example temperatures of about 30° C. to about50° C., for example 30° C., 35° C., 37° C., 40° C., 45° C. or 50° C.,and/or salt concentrations of about 200 mM to about 1000 mM NaCl or theequivalent ionic strength, for example 200 mM, 250 mM, 300 mM, 400 mM,500 mM, 750 mM or 1000 mM. The stringency conditions are dependent onthe length of the nucleic acid and the base composition of the nucleicacid and can be determined by techniques well known in the art. Those ofskill in the art are familiar with these conditions and ranges which areuseful. Generally, a substantially homologous nucleotide sequence is atleast about 75% homologous to SEQ ID NO: 1 or a fragment or subunitthereof, preferably at least about 85% homologous, and most preferably90%, 95% or 99% homologous or more.

Those of skill in the art are familiar with the degeneracy of thegenetic code, and thus are aware that nucleic acid sequences may be lessthan 100% homologous and yet encode the same protein or peptidesequence. Such variation in any of the sequences, fragments, subunits orsubstantially homologous variants also are contemplated as part of thisinvention.

Peptide and protein sequences which are encompassed by this inventioninclude any sequences encoded by the qsbA gene, or any fragment, subunitor substantially homologous variant thereof. Such sequences thereforeinclude any functional protein or peptide which retains the ability toinactivate AHL, including protein and peptide fragments of the completeQsbA protein, such as, for example, the sequences of amino acids 36-217and 233-794 encoding by SEQ ID NO: 1 and substantially homologousvariants thereof. A substantially homologous variant of the QsbA proteinincludes sequences which are at least about 50% homologous, preferablyat least about 60% homologous, and most preferably 70%, 80% or 90%homologous or more. Therefore, a protein which is a substantiallyhomologous varient of QsbA is about 50% to about 99.9% homologous withQsbA. Both conservative and non-conservative amino acid substitutionsare contemplated, as well as sequences containing non-traditional ormodified amino acids such as those known in the art.

The term “fragment” is intended to indicate any portion of thenucleotide of SEQ ID NO: 1 or protein/peptide sequence of SEQ ID NO: 2which is greater than about 300 nucleotide bases or about 100 aminoacids, up to one nucleotide or amino acid less than the entire sequence.The term “subunit” is intended to encompass any functional unit of theQbsA protein, such as, for example, amino acids 36-217 or 233-794 of SEQID NO: 2.

A protein or peptide sequence which is considered to inactivate N-acylhomoserine lactones is one which is capable of inactivating at least 55pmoles N-acyl homoserine lactone (OOHL) per μg protein per minute at 30°C.

It has been previously demonstrated that quenching bacterial quorumsensing by inactivation of N-acyl homoserine lactone with AHL-lactonasestops bacterial infection (9, 10). The gene and protein described here,which is likely an AHL-acylase, represent a new and effective tool forinactivation of AHL signals and thus control bacterial infection.Similarly, the gene and protein described here targets AHLquorum-sensing signals that regulate expression of pathogenic genes ofmany bacterial pathogens at a threshold concentration. This tool isapplicable to all plant, animal or human diseases where the expressionof pathogenic genes of bacterial pathogens is activated by AHL signals,such as, for example, plant pathogens Erw. carotovora, Erw.Chrysanthemi, Erw. Stewartii; human pathogens P. aeruginosa, B. cepacia;and animal pathogens X. nematophilus, P. fluorescens (1, 3, 6, 12, 17,19, 22, 23, 24, 26).

REFERENCES

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Costa and Loper, “EcbI and EcbR: homologs of LuxI and LuxR    affecting antibiotic and exoenzyme production by Erwinia carotovora    subsp. betavasculorum,” Can. J. Microbiol. 43: 1164-1171, 1997.-   7. Daumy et al., “Role of protein subunits in Proteus retigeri    penicillin G acylase,” J. Bacteriol. 163: 1279-1281, 1985.-   8. Davies et al., “The involvement of cell-to-cell signals in the    development of a bacterial biofilm,” Science 280: 295-298, 1998.-   9. Dong et al., “AiiA, an enzyme that inactivates the acyl    homoserine lactone quorum-sening signal and attenuates the virulence    of Erwinia carotovora,” Proc. Natl. Acad. Sci. USA 97: 3526-3531,    2000.-   10. Dong et al., “Quenching quorum sensing-dependent bacterial    infection by an N-acyl homoserine lactonase,” Nature 411: 813-817,    2001.-   11. Dumenyo et al., “Genetic and physiological evidence for the    production of N-acyl homoserine lactones by Pseudomonas syringae pv.    syringae and other fluorescent plant pathogenic Pseudomonas    species,” Eur. J. Plant Pathol. 104: 569-582, 1998.-   12. Dunphy et al., “A homoserine lactone autoinducer regulates    virulence of an insect-pathogenic bacterium, Xenorhabdus    nematophilus (Enterobacteriaceae),” J. Bacteriol. 179: 5288-5291,    1997.-   13. Eberhard et al., Structural identification of autoinducer of    Photobacterium fischeri luciferase,” Biochemistry 20: 2444-2449,    1981.-   14. Eberl et al., “Involvement of N-acyl-L-homoserine lactone    autoinducers in controlling the multicellular behaviour of Serratia    liquefaciens,” Mol. Microbiol. 20: 127-136, 1996.-   15. Fuqua and Winans, “Conserved cis-acting promoter elements are    required for density-dependent transcription of Agrobacterium    tumefaciens conjugal transfer genes,” J. Bacteriol. 178: 435-440,    1996.-   16. Inokoshi et al., “Cloning and sequencing of the aculeacin A    acylase-encoding gene from Actinoplanes utahensis and expression in    Streptomyces lividans,” Gene 119: 29-35, 1992.-   17. Jones et al., “The Lux autoinducer regulates the production of    exoenzyme virulence determination in Erwinia carotovora and    Pseudomonas aeruginosa,” EMBO J. 12:2477-2482, 1993.-   18. Leadbetter and Greenberg, “Metabolism of acyl-homoserine lactone    quorum sensing signals by Variovorax paradoxus,” J. Bacteriol. 182:    6921-6926, 2000.-   19. Lewenza et al., “Quorum sensing in Burkholderia cepacia:    identification of the LuxRI homologs CepRI,” J. Bacteriol. 181:    748-756, 1999.-   20. Matsuda and Komatsu, “Molecular cloning and structure of the    gene for 7β-(4-carboxybutanamido) cephalosporadic acid acylase from    a Pseudomonas strain,” J. Bacteriol. 163: 1222-1228, 1985.-   21. Matsuda et al., “Nucleotide sequence of the genes for two    distinct cephalosporin acylases from a Pseudomonas strain,” J.    Bacteriol. 169: 5821-5826, 1987.-   22. Nasser et al., “Characterization of the Erwinia chrysanthemi    expl-expR locus directing the synthesis of two N-acyl-homoserine    lactone signal molecules,” Mol. Microbiol. 29: 1391-1405, 1998.-   23. Passador et al., “Expression of Pseudomonas aeruginosa virulence    genes requires cell-to-cell communication,” Science 260: 1127-1130,    1993.-   24. Pearson et al., “Structure of the autoinducer required for    expression of Pseudomonas aeruginosa virulence genes,” Proc. Natl.    Acad. Sci. USA 91: 197-201, 1994-   25. Piper et al., “Conjugation factor of Agrobacterium tumefaciens    regulates Ti plasmid transfer by autoinduction,” Nature 362:    448-450, 1993.-   26. Pirhonen et al., “A small diffusible signal molecule is    responsible for the global control of virulence and exoenzyme    production in the plant pathogen Erwinia carotovora,” EMBO J. 12:    2467-2476, 1993.-   27. Schumacher et al., “Penicillin acylase from E. coli: unique    gene-protein relation,” Nucleic Acids Res. 14: 5713-5727, 1986.-   28. Staskawicz et al., “Molecular characterization of cloned    avirulence genes from race 0 and race 1 of Pseudomonas syringae pv.    glycinea,” J. Bacteriol. 169: 5789-5794, 1987.-   29. Takeshima et al., “A deacylation enzyme for aculeacin A, a    neutral lipopeptide antibiotic, from Actinoplanes utahensis:    purification and characterization,” J. Biochem. 105: 606-610, 1989.-   30. Verhaert et al., “Molecular cloning and analysis of the gene    encoding the thermostable penicillin G acylase from Alcaligenes    faecalis,” Appl. Env. Microbiol. 63: 3412-3418, 1997.-   31. Zhang et al., “Agrobacterium conjugation and gene regulation by    N-acyl-L-homoserine lactones,” Nature 362: 446-447, 1993.

The following examples are provided to illustrate the inventiondescribed herein and should not be construed to limit the appendedclaims.

EXAMPLES Example 1 Bacterial Isolation

A bacterial biofilm sample was collected from a water treatment systemand screened to isolate AHL inactivation bacterial strains. Thebacterial mixture was suspended in sterilized water with shaking for 1hour before spreading onto YEB medium (yeast extract, 5 g/l; caseinhydrolysate, 10 g/l; NaCl, 5 g/l; sucrose, 5 g/l; MgSO₄7H₂O, 0.5 g/l andagar, 15 g/l) plates. Individual colonies were restreaked on new platesto ensure purity of the isolates. Bacterial isolates were cultured in LBmedium (tryptone, 10 g/L; yeast extract, 5 g/L, and NaCl, 10 g/L; pH7.0)in 1.5-ml Eppendorf™ tubes or 96-well plates at 28° C., with shaking,overnight, and assayed for AHL inactivation activity.

Example 2 AHL Inactivation Bioassay

The bacterial culture to be assayed was mixed with an equal volume offresh medium containing 20 μM N-β-oxooctanoyl-L-homoserine lactone(OOHL), or another AHL, when specified, to form a reaction mixture. Thereaction mixture was incubated at 28° C. for 4 to 5 hours, followed by30 minute sterilization under UV light. Plates containing 20 ml MM agarmedium (K₂HPO₄₁ 10.5 g/L; KH₂PO₄, 4.5 g/L; MgSO₄.7H₂O, 0.2 g/L; FeSO₄,4.5 mg/L; CaCl₂; 10 mg/L; MnCl₂, 2 mg/L; (NH₄)₂SO₄, 2.0 g/L; mannitol,2.0 g/L; pH 7.0) supplemented with 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (X-Gal, 40 μg/ml) were prepared. The solidifiedmedium was cut, still inside the plate, into separated slices(approximately 1 cm in width). See FIG. 1. Five microliters ofsterilized reaction mixture was loaded at the top of an MM agar strip,and then AHL indicator cells (Agrobacterium tumefaciens strain NT1(traR; tra:lacZ749) (25) 0.7 μl cell suspension with an optical densityat 600 nm of 0.4) were spotted at progressively further distances fromthe loaded samples. Plates were incubated at 28° C. for 24 hours. Apositive result for AHL inactivation is shown by the absence of bluecolonies on the slice. A negative result for AHL inactivation is shownby the presence of blue colonies on the slice. For assay of protein forenzyme activity, total soluble bacterial protein was incubated with 20μM of OOHL (or other AHL) at 37° C. for 1 hour as the reaction mixture.

Example 3 Identification and Cloning the qsbA Gene

Two bacterial isolates from the biofilm sample with distinct phenotypes,XJ12B and XJ12A, were found to possess the ability to inactivate AHL,with XJ12B showing stronger enzyme activity. The XJ12B late wascultured, centrifuged and sonicated. The strongest enzymatic activitywas associated with the cell debris fraction rather than the solubleprotein and supernatant fractions. These results indicated that the AHLinactivation activity is membrane associated. Sequencing of 16S rRNA wasperformed to identify the XJ12A and XJ12B lates. The 16S rRNA sequencesof these isolates showed 97% and 96% identity, respectively, with thatof Ralstonia eutropha.

To identify the gene encoding for AHL inactivation, a cosmid library of1600 clones was constructed in E. coli with the genomic DNA of Ralstoniasp. strain XJ12B. Genomic DNA from the isolated Ralstonia sp. strainXJ12B was partially digested with Sau3A. The resulting DNA fragmentswere ligated to the dephosphorylated BamH1 site of cosmid vector pLAFR3(28). The ligated DNA was packed with Gigapack IIIXL Packaging Extract(Stratagene) and transfected into E. coli DH5alpha. These E. colitransfectants were screened for AHL inactivation activity according tothe methods described in Example 2 using OOHL as the substrate. Only asingle clone (p13H10) was identified as showing AHL inactivationactivity (see FIG. 1A, slice 1). To subclone the gene encoding thedetected activity, cosmid DNA from the positive clone p13H10 waspartially digested with Sau3A and fused into BamH1 digested cloningvector pGEM-3Zf (+). The plasmids were ligated and transformed into E.coli, and the E. coli were assayed for the ability to inactivate AHL asdescribed in Example 2. Clone p2B10, which contains a 4 kb insert, hadAHL inactivation activity (see FIG. 1A, slice 2). The TGS™ TemplateGeneration System F-700 (Finnzymes OY) was used to mutate p2B10 plasmidDNA by randomly inserting the artificial Mu transposon, following themanufacturer's instructions. Plasmid clone p2B10, which contains the 4kb insert containing the qsbA gene, was used as a template. Fifteenmutant clones were produced, and none was able to inactive AHL.Bacterial cultures of E. coli DH5α containing pMUG3 and pMUC6 are shownas examples in FIG. 1A, slices 3 and 4, respectively. Plasmids weresubsequently purified for sequencing using primers supplied in the kit.

Example 4 Sequencing and Sequence Analysis of the qsbA Gene

Sequencing was performed according to known methods using ABI PrismdRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin-ElmerApplied Biosystems). The 4 kb fragment from clone p2B10 was completelysequenced and is shown in Table I. The sequence contains an open readingframe of 2385 nucleotides with an ATG start codon and a TGA stop codon(SEQ ID NO: 1, nucleotides 1259-3643). Based on the MU transposonmutagenesis data described in Example 3, this open reading frame is thecoding region of the AHL inactivation gene, designated as qsbA. Aputative ribosome binding site (AGGAGA) is located 6 base pairs upstreamof the first ATG start codon (underlined in Table I).

The deduced peptide sequence shows the typical polypeptide primarystructure of aculeacin A acylases (AACs) and penicillin G acylases, withsignal peptide-α subunit-spacer-β subunit organization (16, 30). Thereare four additional potential start codons located 3, 36, 189 and 384downstream from the first ATG. The longest open reading frame encodes794 amino acids, with a predicted molecular weight of 85373 Daltons. Thededuced peptide has 78 strongly basic and strong acid amino acidresidues and a predicted isoelectric point of 7.48. The first 20 aminoacid residues of the assumed open reading frame appear to be a signalpeptide.

The peptide sequence of qsbA deduced from the open reading frame shares40-52% identity with AACs from Deinococcus radiodurans strain R1,Actinoplanes utahensis and a putative acylase from Pseudomonasaeruginosa, The AACs' catalyze deacylation of their substrates. TheseAAC genes are translated as single precursor polypeptide and thenprocessed to the active form of two subunits. By alignment with thepeptide sequences from D. radiodurans strain R1, A. utahensis and P.aeruginosa, Table II, the presumed α and β subunits are located at aminoacid positions 36-217 and 233-794, respectively, with a 15 amino acidspacer between them. QsbA shares less than 28% homology with penicillinG acylase (20) and cephalosporin acylase (21). See Table II. The aminoacid sequence alignment in Table II was analyzed by the Clustal Wprogram available from the European Bioinformatics Institute website(http://www.ebi.ac.uk).

Example 5 Expression of the QsbA Gene

The coding region of the qsbA gene was amplified by PCR using a forwardprimer 5′-CGTGGATCCATGATGCAGGATTCGCCGCTGCGC-3′ (SEQ ID NO: 6) and areverse primer 5′-CGCGAATTCACCGGCAGCCCTCATGCGACAAC-3′ (SEQ ID NO: 7)containing BamH1 and EcoR1 restriction sites, respectively. Theamplified PCR products were digested using the above restriction enzymesand fused in-frame to the coding sequence of the glutathioneS-transferase (GST) gene under the control of the isopropylβ-D-thiogalactopyranoside (IPTG) inducible lac promotor in vectorpGEX-2T (Amersham Pharmacia) to generate construct pGST-QsbA. pGST-QsbAwas transformed into E. coli and expressed.

Total soluble protein was extracted from the recombinant E. coli cellsharboring the GST-QsbA-encoding fusion construct according to methodsknown in the art, based on the methods described in Dong et al. (9), andassayed for AHL inactivation. The total soluble protein from E. colicontaining GST vector only was used as a control. For the bioassay, 50μl of the soluble protein preparation (20 μg/μl) was added to the samevolume of 40 μM AHL, e.g., OOHL. After a 1 hour incubation at 37° C.,the reaction mixture was assayed as described in Example 2.Representative data, shown in FIG. 1B, slice 1, indicate that thesoluble GST-QsbA fusion protein effectively eliminated OOHL activity.

Example 6 Characterization of the Substrate Spectrum of GST-QsbA FusionProtein Expressed in E. coli

To determinate the substrate spectrum of QsbA, total soluble proteinextracted from the recombinant E. coli (pGST-QsbA) was assayed forinactivation of AHLs with acyl chains of differing lengths according tothe methods of Example 2. The following AHLs were synthesized accordingto known methods as described by Zhang et al. (31): (1)N-octanoyl-L-homoserine lactone (C8HSL, OOHL); (2)N-decanoyl-L-homoserine lactone (C1DHSL, DHL); (3)N-β-oxohexanoyl-L-homoserine lactone (3-oxo-C6HSL, OHHL); (4)N-β-oxohexanoyl-L-homoserine lactone (3-oxo-C12HSL, OdDHL); (5)N-β-oxohexanoyl-L-homoserine lactone (3-oxo-C8HSL, OOHL). The butyl andhexyl esters of N-β-oxohexanoyl-L-homoserine were prepared byesterification of N-β-oxohexanoyl-L-homoserine lactone with 1-butanoland 1-hexanol respectively, in the presence of small amount of Dowex50H+ resin (Aldrich). The reaction was conducted at 60° C. for 2 hoursand the products were purified by silica column chromatography.

QsbA completely inactivated OOHL, N-β-oxodecanoyl-L-homoserine (ODHL)and N-β-oxododecanoyl-L-homoserine (OdDHL), which have acyl chains of 8,10 and 12 carbons, respectively, at the concentrations tested (data notshown). QsbA also strongly inactivated N-β-octanoyl-L-homoserine (OHL)and N-β-decanoyl-L-homoserine (DHL), which have acyl chains of 8 and 10carbons, respectively (data not shown). However, under the same reactionconditions, QsbA had less inactivating activity forN-β-oxohexanoyl-L-homoserine (OHHL), which has an acyl chain of 6carbons (data not shown). The total soluble protein extract from controlE. coli (pGST) did not show any activity against AHLs (data not shown).

QsbA also completely inactivated the butyl and hexyl esters ofN-β-octanoyl-L-homoserine (data not shown). These two esters ofN-β-octanoyl-L-homoserine showed comparable induction activity with OOHLwhen assayed with the AHL reporter strain A. tumefaciens NT1 (traR;tra:lacZ749) (data not shown). AHL-lactonase (encoded by aiiA) did notinactivate these substrates. These substrate specificity data areconsistent with identification of QsbA as an AHL-acylase.

Example 7 Purification of AHL-Acylase Encoded by the qsbA Gene

The GST-[AHL-acylase] fusion protein was purified using a glutathioneSepharose 4B affinity column following the manufacturer's instructions(Pharmacia). AHL-acylase was cleaved by digestion with thrombin (Sigma).Protein concentration was determined by measuring OD₂₈₀.

The purified AHL-acylase was incubated with OOHL for 20 minutes and therelative enzyme activity was measured by determining the residual OOHLin the reaction mixture, which contained 80M OOHL and about 0.6 μgAHL-acylase in a total reaction volume of 50 ml 1×PBS buffer. Thereactions were stopped by addition of 1% SDS before loading on the assayplate. Determination of the OOHL activity was carried out inquadruplicate. AHL-acylase degraded OOHL in a range of temperatures from22-42° C. at pH 7.0. See FIG. 2. The optimal temperature for enzymeactivity was found to be approximately 28° C. Reaction temperaturehigher than 42° C. decreased enzyme activity sharply. The optimal pH forenzyme activity also was determined. The AHL-acylase has a relativelynarrow optimal pH range from pH 6.5 to 7.5. See FIG. 2. The time courseof OOHL inactivation by the purified AHL-acylase was determined at 30°C. See FIG. 3. After 10 minutes, more than 82% OOHL had been degraded;the reaction rate was estimated to be about 55 pmols per μg AHL-acylaseper minute.

1. A composition of matter which comprises an isolated nucleic acidaccording to SEQ ID NO:
 1. 2. A composition of matter which comprises anisolated nucleic acid which comprises nucleotides 1234-3618 of SEQ IDNO:
 1. 3. A composition of matter which comprises an isolated peptidicsequence encoded by a nucleic acid consisting of nucleotides 1234-3618of SEQ ID NO:
 1. 4. A composition of matter which comprises an isolatedpeptidic sequence according to SEQ ID NO:
 2. 5. A composition of matterwhich comprises an isolated peptidic sequence comprising amino acids36-217 of SEQ ID NO:
 2. 6. A composition of matter which comprises anisolated peptidic sequence comprising amino acids 233-794 of SEQ ID NO:2.
 7. A composition of matter according to claim 4 which inactivatesAHL.
 8. A method of modulating AHL signaling activity which comprisescontacting said AHL with a composition of matter according to any one ofclaims 3 or 4-7.
 9. A transgenic plant harboring a nucleic acid of claim2.
 10. A transgenic non-human animal harboring a nucleic acid of claim2.
 11. A method of controlling a bacterial disease in a mammal in needthereof which comprises administering to said mammal a composition ofmatter according to any one of claims 3 or 4-7, wherein the expressionof pathogenic genes of said bacteria are regulated by AHL signals.
 12. Amethod of claim 12 wherein said mammal is a human.
 13. A method ofcontrolling a bacterial disease in a plant in need thereof whichcomprises administering to said plant a composition of matter accordingto any one of claims 3 or 4-7, wherein the expression of pathogenicgenes of said bacteria are regulated by AHL signals.
 14. A method ofcontrolling a bacterial disease in a mammal in need thereof whichcomprises administering to said mammal a composition of matter of claim2 and its peptide product, wherein the expression of pathogenic genes ofsaid bacteria are regulated by AHL signals.
 15. A method of claim 14wherein said mammal is a human.
 16. A method of controlling a bacterialdisease in a plant in need thereof which comprises administering to saidplant a composition of matter of claim 2, wherein the expression ofpathogenic genes of said bacteria are regulated by AHL signals.
 17. Amethod of controlling a bacterial disease in a plant using any bacterialspecies containing the composition of matter of claim 2.